This application claims foreign priority of Italy VR98A000033, filed Apr. 30, 1998.
The present invention relates to a method for production on the spot of a peracetic acid (PAA) aqueous solution (disinfectant system) used for high-level chemical disinfection and chemical cold-sterilization of many devices, equipment and plants, e.g.
medical and surgical devices, including fiber-optic instruments (endoscopes, etc.), bed sheets or other fabrics used in the sanitary field (hospitals, surgeries, etc.);
tools, surfaces, instruments, CIP (cleaning in place) pipes, and objects in general in the field of food handling;
wastewater treatment plants;
and in any other field requiring a high-level disinfection or chemical cold-sterilization treatment.
Efficient sterilization methods are required for industrial and sanitary applications. For their repeated use, various tools and devices require safe, effective and fast disinfection and sterilization procedures. Although most xe2x80x9ccriticalxe2x80x9d multiple-use medical-surgical instruments are sterilized, after accurate cleaning, by:
dry heat treatment,
wet heat treatment (steam autoclave),
ethylene oxide sterilization (for plastics),
there are, however, devices (for example endoscopes, etc.), especially in the sanitary field, which are made of highly heat-sensitive material and thus cannot be subjected to the above treatments.
Moreover, since some of such devices are often used for diagnostic and therapeutic purposes in daily activity, their passage through ethylene oxide autoclaves, although being feasible, is impractical owing to both excessively high costs and time limits. Accordingly, high-level disinfection and sterilization with chemical products at room temperature is the only feasable procedure for such instruments.
Many xe2x80x9ccold-sterilizingxe2x80x9d compositions have been suggested so far, e.g. 2%, 2.5%, 3.2% glutaraldehyde, buffered to pH 7.5-8.5 upon being used, and the phenol-phenate buffer system in association with glutaraldehyde (Sporicidin(trademark)) or, more recently, xe2x80x9cSporicidin Plus(trademark)xe2x80x9d, an association of phenol, phenolic derivatives and glutaraldehyde; see U.S. Pat. No. 4,103,001 (Schattner). All these products have in common the presence of glutaraldehyde and make it possible to obtain sterilization of the devices, but only after extremely long contact times.
A process or a product can be considered to be a sterilizer only if it can eliminate all microbial life forms, including spores, which have the highest resistance to sterilization processes. Accordingly, a sterilizing chemical preparation must be bactericidal, fungicidal, virucidal and sporicidal.
A relatively small number of antimicrobial agents is actually sporicidal and thus usable as xe2x80x9cchemosterilizersxe2x80x9d. One of them is peracetic acid, a peroxide agent, which has a wide and rapid germicidal activity. The FDA (Food and Drug Administration) has long recognized as 510 (k)-cleared chemical sterilization agents two products whose active principle is peracetic acid:
Steris 20(trademark) (0.2% peracetic acid at 50-56xc2x0 C. for 12 minutes of contact)
Peract 20(trademark) (0.08% peracetic acid plus 1% hydrogen peroxide at 20xc2x0 C. for 8 hours of contact).
The first product is a concentrated solution of 35% w/w peracetic acid, which has a six-month stability period. The second product is a ready-for-use acid aqueous solution, which has a stability period of one year.
Peracetic acid has the same attributes as hydrogen peroxide (germicidal and sterilizing capacities, non-dangerous decomposition products and infinite solubility in water), but is more soluble in lipids and insensitive to deactivation by catalase and peroxidase enzymes. It is also a more powerful antimicrobial agent than hydrogen peroxide, since it is rapidly active at low concentrations against a broad range of microorganisms. Furthermore, it is sporicidal at very low temperatures and remains effective even in the presence of organic material. As a weak acid it is more active in an acid environment.
Aqueous solutions of concentrated peracetic acid and hydrogen peroxide have already been proposed commercially; however, they have a very short stability period. A certain stability is ensured by the presence of an excess of acetic acid and/or hydrogen peroxide with respect to the equilibrium for solutions of peracetic acid ranging from 0.5% to 50% w/w. Moreover, the addition of a sequestrant for metallic ions to the aqueous solution, e.g. a diphosphonic acid and an anionic surfactant belonging to the class of alkylbenzene sulfonates, alkylsulfonates or alkane sulfonates, as disclosed in U.S. Pat. No. 4,051,058 (Bowing et al.), ensures further stability of the concentrated peracetic-acid aqueous solutions used to prepare diluted microbicidal solutions in a sanitary and food-handling environment. However, these concentrated solutions, when correctly preserved, can be considered as stable for up to six months without appreciable losses of active oxygen.
The xe2x80x9csix monthxe2x80x9d term is highly penalizing from the commercial point of view, especially because it does not allow storage on an industrial scale (that is to say, storage in relevant amounts), and this has a negative effect on the final cost of peracetic acid (PAA).
In general, peroxides are high energy state compounds and as such can be considered to be thermodynamically unstable. Peracetic acid (PAA) is much less stable than hydrogen peroxide (HP). A 40% w/w solution of PAA loses 1 to 2% of its active ingredient per month, compared with hydrogen peroxide (30 to 90%), which loses less than 1% per year. The decomposition products of peracetic acid are acetic acid, hydrogen peroxide (HP), oxygen and water. Dilute solutions of peracetic acid are even less stable. Thus, for example, a 1% solution loses half of its concentration by hydrolysis in six days.
After succeeding in producing highly concentrated hydrogen peroxide, peracetic acid has been produced on an industrial scale by the reaction of acetic acid or acetic anhydride with concentrated hydrogen peroxide in the presence of sulfuric acid, which acts as a catalyst, as shown by the following reaction formula: 
In order to prevent the inverse reaction, peracetic acid solutions are boosted with acetic acid and hydrogen peroxide (HP). Moreover, a stabilizing agent is used, e.g. a sequestrant (sodium pyrophosphate) or a chelating agent (8-hydroxyquinoline) to remove any trace of metallic ions, which would accelerate the decomposition of peroxides. A system in which use is made of anionic surfactants in PAA solutions has not only higher stability but also a higher antimicrobial activityxe2x80x94see U.S. Pat. No. 4,051,058. Finally, synergistic effects between PAA and ethyl alcohol or isopropyl alcohol have been noted in connection with germicidal activity.
Despite the various proposals as described above, when stable commercial peracetic acid is diluted with water it decomposes rapidly. Such a decomposition can be accelerated by high temperatures and the presence of heavy metals in the solution. Accordingly, it is advisable to dilute the peracetic acid with deionized or distilled water, store it in a cool place and use it as soon as possible. The decomposition of peracetic acid can occur in the three following manners:
Oxygen is formed in reaction (1). The rate of decomposition in this way depends on the nature and quantity of heavy metals in the solution. This reaction is often responsible for the loss of activity of insufficiently stabilized PAA solutions. 
Reaction (2) proceeds via intermediate radicals. Methyl radicals may be formed among other substances. Similarly to reaction (1), also this reaction is catalyzed by metallic ions.
The hydrolysis of peracetic acid (reaction 3) is highly pH-dependent. It takes place whenever peracetic acid solutions are diluted. The reaction products are acetic acid and hydrogen peroxide.
Every dilution of commercially available PAA solutions results in a new equilibrium being reached between peracetic acid, acetic acid, hydrogen peroxide and water. Although it is possible to dilute solutions of peracetic acid, the concentration obtained upon dilution may change later because the equilibrium can shift.
Peracetic acid is reactive in any concentration and can only be stored for long periods if adequately stabilized. In controlled storage conditions and at room temperature the loss of activity is very small. Peracetic acid has a high oxidation potential and is highly reactive. As the pH increases, its stability decreases. When it is put contact with sodium hydroxide or other alkaline media, decomposition occurs. Ions of heavy metals, e.g. copper, manganese and iron, start catalytic decomposition. Heat and contact with incompatible solutions might give rise to decomposition process.
The main object of the present invention is to provide a simple system for preparing on demand a sterilizing aqueous solution based on peracetic acid and other additives that enhance its germicidal properties with a synergistic effect. Since this system generates peracetic acid only upon being used, it not affected by stability and storage problems as mentioned above, which are due to its active principle and faced with all commercially available aqueous solutions of various concentrations of peracetic acid.
This and other objects which will become better apparent hereinafter are achieved, according to the invention, by a two-component system which comprises a main parent solution and an activator to be added to the parent solution immediately before use to form peracetic acid by simple equilibrium.
Advantageously, to ensure higher stability, germicidal activity and compatibility with the treated materials, preparation on the spot before use is made at room temperature and the solution can be buffered to a pH ranging from 3.5 to 7.0 at 20xc2x0 C. Moreover, such a system according to the invention is a new solution of peracetic acid, which differs from commercially available solutions since it has, at equilibrium, a variable excess of one of the two reagents to avoid hydrolysis phenomena which affect the active principle. As a matter of fact, concentrated peracetic acid solutions available on the market have a very specific concentration of hydrogen peroxide and acetic acid in excess with respect to the equilibrium. Thus, regardless of the dilution, molar ratios among the various components remain always the same.
According to the present invention, since concentration of the acetylated compound (acetic acid or tetraacetyl glycoluryl) in the parent solution and concentration of the peroxide in the activator can vary independently from one another, it is possible to obtain a wide variety of concentrations in excess with respect to equilibrium of one or both reactive components in the activated solution.
Furthermore, concentrations of peracetic acid that can be obtained from the preparation according to the present invention can vary between 0.01% and 10.00% w/w. With this concentration range, it is possible to achieve a wide range of ready-for-use solutions and of concentrated solutions to be diluted in water, having a high bactericidal, fungicide, virucidal, tubercolicidal and sporicidal activity for extremely short contact times.
A. The parent solution has the following composition:
and optionally at least one of the following components:
1. An acetate salt, in a variable amount depending upon the % of acetic acid in order to form the buffer system for buffering the pH to 3.50-7.00 at 20xc2x0 C.;
2. Other buffer systems suitable for buffering the pH of the parent solution and the activated solution at between 3.5 and 7.0 at 20xc2x0 C. (e.g. sodium carbonate, sodium bicarbonate, monosodium phosphate, sodium hydroxide; weak monocarboxylic and polycarboxylic organic acids such as citric acid, tartaric acid or the like);
3. A oxide reduction indicator (such as amaranth red) as necessary (traces)
Essential and indispensable reactive components of the parent solution are O-acetylated compounds, such as glacial acetic acid, and N-acetylated compounds, such as tetracetyl glycoluryle (TAGU) or tetraacetyl ethylene diamine (TAED).
B. The activator comprises a single component, i.e. a peroxide compound, such as potassium peroxydisulfate, peroxymonosulfate, peroxydisulphonic acid, hydrogen peroxide and derivatives thereof, e.g. potassium and sodium perborates, carbamide peroxide (also known as urea hydrogen peroxide), all of them in any physical state (solid, liquid and vapor) and at different concentrations of active oxygen. The amount of activator to be added to the parent solution can vary according to the desired concentration of peracetic acid and the molar excess of hydrogen peroxide with respect at equilibrium with respect to the other reagent (N- or O-acetylate derivative).
The present invention provides for a two-component system for preparation on the spot of a sterilizing aqueous peracetic acid solution and other additives which enhance its stability and germicidal activity as well as compatibility with various materials. These solutions can be concentrated and therefore intended for dilution with water just before use, or can be solutions ready for use. They can have an acid pH or a buffered pH in the range of 3.50 to 7.00 at 20xc2x0 C. for obtaining better compatibility with the materials. The two components are stored separately in two separate packages and combined only immediately before use. Peracetic acid forms only upon combination through chemical-physical equilibrium. In this manner, any problem related to the instability of peracetic acid and therefore to its extended storage are eliminated, since active principle forms only after combining two separately stored components.
This system makes also possible to obtain a solution which has markedly better germicidal, stability and materials-compatibility characteristics than conventional aqueous solutions based on PAAxe2x80x94see the following
For a better understanding of the innovative characteristics according to the present invention one should consider the function of each component.
A. Parent solution
a) N-acetylated compounds (tetracetyl glycoluryle (TAGU) or tetracetyl ethylene diamine (TAED): this is the essential reagent for the preparation of PAA by reaction with peroxide activator (see formula 4). It has the same function as acetic acid. However, with respect to acetic acid it reacts more quickly with hydrogen peroxide. In terms of reaction kinetics, one could say that tetraacetyl glycoluryle is more electrophilic than acetic acid. Said N-acetylated derivative, therefore, allows peracetic acid to be rapidly formed when the parent solution is combined with activator. This reaction does not even require a catalyst. It is in fact known that nucleophilic attack of carbonyl carbon occurs more quickly for an imide (such as tetraacetyl glycoluryle) and an amide than for an acid or an ester. Chlorides of acids and anhydrides are even more electrophilic, but due to their high reactivity and instability in an aqueous solution they cannot be used for this purpose. 
b) Acetic acid has the same function as tetraacetyl glycoluryle, the only difference being that its reaction kinetics with hydrogen peroxide, other conditions being equal, is slower. 
Accordingly, within the formulation the presence of tetraacetyl glycoluryle and of acetic acid allows immediate and prolonged formation of peracetic acid (PAA) as a consequence of activation. By varying their concentration, both the concentration at equilibrium of PAA and the reaction kinetics change. In other words, both the percentage w/w of peracetic acid and the waiting period after activation vary to obtain a preset concentration of PAA and the duration of the solution.
c) Acetated salts, e.g. sodium acetate, are the conjugate salts of a weak acid. Accordingly, together with the acid a buffer system is formed which buffers the pH of the solution to a value close to its own value of pKa, which is 4.75 in this case. Buffering the solution to these values of pH is a further innovation with respect to conventional solutions of PAA, which are highly acid. As a matter of fact, this makes it possible to use the solution also to sterilize particularly delicate instruments, such as fiber-optic instruments, without the risk of causing corrosive effects, which are much more frequent with highly acid solutions.
d) As a buffer system, it is possible to use all weak organic acids, as specified above, and the corresponding conjugate salts, or any strong base which allows an acetic acid/acetate salt buffer system to be generated. There are in fact two ways for buffering the pH:
by adding, simultaneously with the solution to be buffered, a weak acid and, in a stoichiometric quantity, the corresponding conjugate salt;
by adding only a weak acid and a strong base which is capable of converting part of the acid into its conjugate base or, vice versa, by adding the conjugate salt and a strong acid in order to convert part of the salt into the corresponding conjugate weak acid.
e) The oxyde-reduction indicator is designed to indicate to the operator that the parent solution has been activated. This is a compound which has a different color in the reduced state and in the oxidized state. By adding the activator, the oxidation state of the solution environment varies and accordingly the solution acquires a different color.
f) The primary, secondary and tertiary alcohols that can be used are short-chain alcohols, such as ethyl alcohol, n-propyl alcohol, isopropyl alcohol, butyl alcohol, or medium/long-chain alcohols, such as undecyl alcohol and the like, alkyl or arylalkyl alcohols having a germicidal activity. Of course, the alcohols that can be used in the composition according to the present invention must have a more or less significant miscibility with water to be able to mix uniformly with water without producing phase separation or a more or less stable emulsion. The presence of such an element constitutes an innovation with respect to all other disinfectant solutions based on PAA that are currently commercially available. The alcohol, in addition to acting as a preservative for the parent solution, in fact, also has a germicidal synergistic effect with the PAA.
g) The stabilizing agents are sequestrants or chelating agents (ethylenediaminotetraacetic acid (EDTA) and salts thereof, sodium tripolyphosphate, sodium pyrophosphate), which are designed to remove the traces of metallic ions, which would accelerate the decomposition of the peroxides. This category of stabilizing agents also includes the phosphonic acids disclosed in U.S. Pat. No. 4,051,058. However, preference is given to phosphonic acids having a low relative molecular mass and containing at least two anionic groups, one of which is a phosphonic acid group. These also include diphosphonic acids having the following formula:
R1CH5O7P2; R1R2R3CH5O6NP2 
where R1 is a member chosen from the group that contains phenyl, cycloalkyl having 5 to 6 carbon atoms and alkyl having 1 to 6 carbon atoms, R2 and R3 are members chosen from the group comprising hydrogen, alkyls having 1 to 4 carbon atoms and amino alkyls having 1 to 4 carbon atoms. These include:
aminotri(methylene phosphonic acid)
dimethylaminomethane diphosphonic acid
aminoacetic acid N-di-(methylene phosphonic acid)
ethylene diamine tetra-(methylene phosphonic acid)
1-amino-1-cyclohexylmethane-1,1-diphosphonic acid
3-aminopropane-1-hydroxy-1,1-diphosphonic acid
2-phosphone butane-1,2,4-tricarboxylic acid
phosphone succinic acid
1-phospone-1-methylsuccinic acid
The following are particularly preferable:
dimethylamine methane diphosphonic acid
1-amino-1-phenyl methane diphosphonic acid
amino-tri-(methylene phosphonic acid)
aminoacetic acid N-di-(methylene phosphonic acid)
1-hydroxyethane-1,1-diphosphonic acid
These acids can also be used in the form of their water-soluble salts, particularly as alkaline metals, such as sodium and potassium. If required, it is also possible to use mixtures of individual phosphonic acids or of acid salts thereof.
h) The presence of at least one anionic surfactant ensures not only higher stability but also higher antimicrobial activity of the diluted solutions of PAA, as disclosed in U.S. Pat. No. 4,051,058. The anionic surfactants that can be used in the composition according to the present invention are of sulfate and sulfonate type, such as alkylbenzene sulfonates having 6 to 18 carbon atoms in the alkyl, alkyl sulfates and/or alkane sulfates (each having 8 to 22 carbon atoms in the alkyl or alkane group), added in amounts between 0.001 and 5% w/w.
The alkyl benzene sulfonates that can be used are preferably those containing an alkyl radical with 6 to 18 carbon atoms, preferably 9 to 15 carbon atoms. Instead of alkyl benzene sulfonates, it is possible to use alkyl sulfates or alkane sulfates with an alkyl or alkane radical having a chain of 12-18 carbon atoms. If required, it is of course possible to use mixtures of the above mentioned anionic surfactant compounds disclosed in U.S. Pat. No. 4,051,058.
i) The wetting agents that can be used belong to the category of polyols, such as propylene glycol, diethylene glycol and glycerine. All these compounds can be mixed with water and are designed to protect the objects to be disinfected from any aggressiveness of the product.
j) The strong inorganic acid acts as a catalyst for the reaction for preparing PAA, reducing the activation energy, i. e. providing a faster reaction. Like all the catalysts, the inorganic acid does not take part to the reaction and thus a few traces in the solution are sufficient. Among strong inorganic acids, preference is given to the use of concentrated sulfuric acid, as it is also used for the same purposes in industrial chemical production of concentrated solutions of peracetic acid. Besides being a strong acid, it is also an oxidizing and dehydrating agent. In this case, however, addition of inorganic acid affects only the rate of formation of the peracetic acid.
k) Purified water constitutes the solvent of the parent solution. The presence of this component is also an innovation, especially with respect to concentrated solutions based on PAA to be diluted at the time of use. The use of tape water for dilution in fact very often compromises the solution stability due to the presence of metallic ions which assist degradation of the peroxides.
B. Activator
The activator is a peroxide compound which releases active oxygen in water. All peroxide compounds share the active function R1xe2x80x94Oxe2x80x94Oxe2x80x94R2, which is responsible for the nucleophilic attack of the carbonyl carbon of the acetyl substrates. More particularly, concentrated hydrogen peroxide or derivatives thereof are used in any physical form and in any concentration. H2O2 (hydrogen peroxide) is the reagent involved in the formation equilibrium of the peracetic acid (PAA). It can be used in the form of an acid solution or as a salt, such as sodium perborate, carbamide peroxide which are instead basic in an aqueous solution.
Activation consists in combining the parent solution with the activator. After a waiting period in the range of between 30 minutes and 48 hours depending on the specific case, PAA concentrations are obtained which have broad-spectrum and swift microbicidal effects. These concentrations and activities remain stable for at least 5 days, if the activated solutions are stored at room temperature and are not subjected to intense shaking. Stability can be extended further if the solution is stored after use in a closed container away from heat and light sources.
Some currently preferred examples of preparation of a xe2x80x9cchemosterilizingxe2x80x9d solution according to the present invention are given hereinafter.